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September 22, 2019  |  

Characterization of phenotypic variation and genome aberrations observed among Phytophthora ramorum isolates from diverse hosts.

Accumulating evidence suggests that genome plasticity allows filamentous plant pathogens to adapt to changing environments. Recently, the generalist plant pathogen Phytophthora ramorum has been documented to undergo irreversible phenotypic alterations accompanied by chromosomal aberrations when infecting trunks of mature oak trees (genus Quercus). In contrast, genomes and phenotypes of the pathogen derived from the foliage of California bay (Umbellularia californica) are usually stable. We define this phenomenon as host-induced phenotypic diversification (HIPD). P. ramorum also causes a severe foliar blight in some ornamental plants such as Rhododendron spp. and Viburnum spp., and isolates from these hosts occasionally show phenotypes resembling those from oak trunks that carry chromosomal aberrations. The aim of this study was to investigate variations in phenotypes and genomes of P. ramorum isolates from non-oak hosts and substrates to determine whether HIPD changes may be equivalent to those among isolates from oaks.We analyzed genomes of diverse non-oak isolates including those taken from foliage of Rhododendron and other ornamental plants, as well as from natural host species, soil, and water. Isolates recovered from artificially inoculated oak logs were also examined. We identified diverse chromosomal aberrations including copy neutral loss of heterozygosity (cnLOH) and aneuploidy in isolates from non-oak hosts. Most identified aberrations in non-oak hosts were also common among oak isolates; however, trisomy, a frequent type of chromosomal aberration in oak isolates was not observed in isolates from Rhododendron.This work cross-examined phenotypic variation and chromosomal aberrations in P. ramorum isolates from oak and non-oak hosts and substrates. The results suggest that HIPD comparable to that occurring in oak hosts occurs in non-oak environments such as in Rhododendron leaves. Rhododendron leaves are more easily available than mature oak stems and thus can potentially serve as a model host for the investigation of HIPD, the newly described plant-pathogen interaction.

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September 22, 2019  |  

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

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September 22, 2019  |  

The complete genome sequence of Vibrio aestuarianus W-40 reveals virulence factor genes.

Vibrio aestuarianus is an opportunistic environmental pathogen that has been associated with epidemics in cultured shrimp Penaeus vannamei. Hepatopancreas microsporidian (HPM) and monodon slow growth syndrome (MSGS) have been reported in cultured P. vannamei. In this study, we sequenced and assembled the whole genome of V. aestuarianus strain W-40, a strain that was originally isolated from the intestines of an infected P. vannamei. The genome of V. aestuarianus strain W-40 contains two circular chromosomes of 483,7307 bp with a 46.23% GC content. We identified 4,457 open reading frames (ORFs) that occupy 86.35% of the genome. Vibrio aestuarianus strain W-40 consists primarily of the ATP-binding cassette (ABC) transporter system and the phosphotransferase system (PTS). CagA is a metabolism system that includes bacterial extracellular solute-binding protein. Glutathione reductase can purge superoxide radicals (O22-) and hydrogen peroxide (H2 O2 ) damage in V. aestuarianus strain W-40. The presence of two compete type I restriction-modification systems was confirmed. A total of 42 insertion sequences (IS) elements and 16 IS elements were identified. Our results revealed a host of virulence factors that likely contribute to the pathogenicity of V. aestuarianus strain W-40, including the virulence factor genes vacA, clpC, and bvgA, which are important for biofilm dispersion. Several bacitracin and tetracycline antibiotic resistance-encoding genes and type VI secretion systems were also identified in the genome. The complete genome sequence will aid future studies of the pathogenesis of V. aestuarianus strain W-40 and allow for new strategies to control disease to be developed.© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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September 22, 2019  |  

A mosaic monoploid reference sequence for the highly complex genome of sugarcane.

Sugarcane (Saccharum spp.) is a major crop for sugar and bioenergy production. Its highly polyploid, aneuploid, heterozygous, and interspecific genome poses major challenges for producing a reference sequence. We exploited colinearity with sorghum to produce a BAC-based monoploid genome sequence of sugarcane. A minimum tiling path of 4660 sugarcane BAC that best covers the gene-rich part of the sorghum genome was selected based on whole-genome profiling, sequenced, and assembled in a 382-Mb single tiling path of a high-quality sequence. A total of 25,316 protein-coding gene models are predicted, 17% of which display no colinearity with their sorghum orthologs. We show that the two species, S. officinarum and S. spontaneum, involved in modern cultivars differ by their transposable elements and by a few large chromosomal rearrangements, explaining their distinct genome size and distinct basic chromosome numbers while also suggesting that polyploidization arose in both lineages after their divergence.

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September 22, 2019  |  

Ring synthetic chromosome V SCRaMbLE.

Structural variations (SVs) exert important functional impacts on biological phenotypic diversity. Here we show a ring synthetic yeast chromosome V (ring_synV) can be used to continuously generate complex genomic variations and improve the production of prodeoxyviolacein (PDV) by applying Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) in haploid yeast cells. The SCRaMbLE of ring_synV generates aneuploid yeast strains with increased PDV productivity, and we identify aneuploid chromosome I, III, VI, XII, XIII, and ring_synV. The neochromosome of SCRaMbLEd ring_synV generated more unbalanced forms of variations, including duplication, insertions, and balanced forms of translocations and inversions than its linear form. Furthermore, of the 29 novel SVs detected, 11 prompted the PDV biosynthesis; and the deletion of uncharacterized gene YER182W is related to the improvement of the PDV. Overall, the SCRaMbLEing ring_synV embraces the evolution of the genome by modifying the chromosome number, structure, and organization, identifying targets for phenotypic comprehension.

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September 22, 2019  |  

Extraordinary genome instability and widespread chromosome rearrangements during vegetative growth

The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on “core” and “accessory” chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro, but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability. Copyright © 2018 by the Genetics Society of America.

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September 22, 2019  |  

Genomic assemblies of newly sequenced Trypanosoma cruzi strains reveal new genomic expansion and greater complexity.

Chagas disease is a complex illness caused by the protozoan Trypanosoma cruzi displaying highly diverse clinical outcomes. In this sense, the genome sequence elucidation and comparison between strains may lead to disease understanding. Here, two new T. cruzi strains, have been sequenced, Y using Illumina and Bug2148 using PacBio, assembled, analyzed and compared with the T. cruzi annotated genomes available to date. The assembly stats from the new sequences show effective improvement of T. cruzi genome over the actual ones. Such as, the largest contig assembled (1.3?Mb in Bug2148) in de novo attempts and the highest mean assembly coverage (71X for Y). Our analysis reveals a new genomic expansion and greater complexity for those multi-copy gene families related to infection process and disease development, such as Trans-sialidases, Mucins and Mucin Associated Surface Proteins, among others. On one side, we demonstrate that multi-copy gene families are located near telomeric regions of the “chromosome-like” 1.3?Mb contig assembled of Bug2148, where they likely suffer high evolutive pressure. On the other hand, we identified several strain-specific single copy genes that might help to understand the differences in infectivity and physiology among strains. In summary, our results indicate that T. cruzi has a complex genomic architecture that may have promoted its evolution.

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September 22, 2019  |  

Genomic insights into virulence mechanisms of Leishmania donovani: evidence from an atypical strain.

Leishmaniasis is a neglected tropical disease with diverse clinical phenotypes, determined by parasite, host and vector interactions. Despite the advances in molecular biology and the availability of more Leishmania genome references in recent years, the association between parasite species and distinct clinical phenotypes remains poorly understood. We present a genomic comparison of an atypical variant of Leishmania donovani from a South Asian focus, where it mostly causes cutaneous form of leishmaniasis.Clinical isolates from six cutaneous leishmaniasis patients (CL-SL); 2 of whom were poor responders to antimony (CL-PR), and two visceral leishmaniasis patients (VL-SL) were sequenced on an Illumina MiSeq platform. Chromosome aneuploidy was observed in both groups but was more frequent in CL-SL. 248 genes differed by 2 fold or more in copy number among the two groups. Genes involved in amino acid use (LdBPK_271940) and energy metabolism (LdBPK_271950), predominated the VL-SL group with the same distribution pattern reflected in gene tandem arrays. Genes encoding amastins were present in higher copy numbers in VL-SL and CL-PR as well as being among predicted pseudogenes in CL-SL. Both chromosome and SNP profiles showed CL-SL and VL-SL to form two distinct groups. While expected heterozygosity was much higher in VL-SL, SNP allele frequency patterns did not suggest potential recent recombination breakpoints. The SNP/indel profile obtained using the more recently generated PacBio sequence did not vary markedly from that based on the standard LdBPK282A1 reference. Several genes previously associated with resistance to antimonials were observed in higher copy numbers in the analysis of CL-PR. H-locus amplification was seen in one cutaneous isolate which however did not belong to the CL-PR group.The data presented suggests that intra species variations at chromosome and gene level are more likely to influence differences in tropism as well as response to treatment, and contributes to greater understanding of parasite molecular mechanisms underpinning these differences. These findings should be substantiated with a larger sample number and expression/functional studies.

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September 22, 2019  |  

3D molecular cytology of Hop (Humulus lupulus) meiotic chromosomes reveals non-disomic pairing and segregation, aneuploidy, and genomic structural variation.

Hop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker segregation. Cytogenetic analysis of meiotic chromosome behavior has also revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including those involving autosomes in late prophase. To explore the role of meiosis in segregation distortion, we undertook 3D cytogenetic analysis of hop pollen mother cells stained with DAPI and FISH. We used telomere FISH to demonstrate that hop exhibits a normal telomere clustering bouquet. We also identified and characterized a new sub-terminal 180 bp satellite DNA tandem repeat family called HSR0, located proximal to telomeres. Highly variable 5S rDNA FISH patterns within and between plants, together with the detection of anaphase chromosome bridges, reflect extensive departures from normal disomic signal composition and distribution. Subsequent FACS analysis revealed variable DNA content in a cultivated pedigree. Together, these findings implicate multiple phenomena, including aneuploidy, segmental aneuploidy, or chromosome rearrangements, as contributing factors to segregation distortion in hop.

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September 22, 2019  |  

Leishmania genome dynamics during environmental adaptation reveal strain-specific differences in gene copy number variation, karyotype instability, and telomeric amplification.

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery. Copyright © 2018 Bussotti et al.

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September 22, 2019  |  

Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic improvement

Sweetpotato [Ipomoea batatas (L.) Lam.] is a globally important staple food crop, especially for sub-Saharan Africa. Agronomic improvement of sweetpotato has lagged behind other major food crops due to a lack of genomic and genetic resources and inherent challenges in breeding a heterozygous, clonally propagated polyploid. Here, we report the genome sequences of its two diploid relatives, I. trifida and I. triloba, and show that these high-quality genome assemblies are robust references for hexaploid sweetpotato. Comparative and phylogenetic analyses reveal insights into the ancient whole-genome triplication history of Ipomoea and evolutionary relationships within the Batatas complex. Using resequencing data from 16 genotypes widely used in African breeding programs, genes and alleles associated with carotenoid biosynthesis in storage roots are identified, which may enable efficient breeding of varieties with high provitamin A content. These resources will facilitate genome-enabled breeding in this important food security crop.

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September 22, 2019  |  

Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species.

Candida auris is an emergent multidrug-resistant fungal pathogen causing increasing reports of outbreaks. While distantly related to C. albicans and C. glabrata, C. auris is closely related to rarely observed and often multidrug-resistant species from the C. haemulonii clade. Here, we analyze near complete genome assemblies for the four C. auris clades and three related species, and map intra- and inter-species rearrangements across the seven chromosomes. Using RNA-Seq-guided gene predictions, we find that most mating and meiosis genes are conserved and that clades contain either the MTLa or MTLa mating loci. Comparing the genomes of these emerging species to those of other Candida species identifies genes linked to drug resistance and virulence, including expanded families of transporters and lipases, as well as mutations and copy number variants in ERG11. Gene expression analysis identifies transporters and metabolic regulators specific to C. auris and those conserved with related species which may contribute to differences in drug response in this emerging fungal clade.

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September 22, 2019  |  

Complete and de novo assembly of the Leishmania braziliensis (M2904) genome.

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.

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September 21, 2019  |  

Recent advances in bioinformatics for fish genomics

In the past few years, we have contributed efforts to ~1/5 of the reported fish genomes. Based on our related experience, here we outline recent advances in bioinformatics for fish genomics, with an emphasis on development of software for genome assembly, genome annotation and evolutionary analysis. This review will be helpful for the new players of genome analysis on both animals and plants. In the past decade, whole genome sequences of approximately 50 fish species have been reported [1]. We have been involved in ~1/5 of these international works from 2014 to 2017, such as mudskippers (2014) [2], Chinese large yellow croaker [3], Chinese barbel fishes [4], Asian arowana [5,6], Channel catfish [7], seahorses [8], Japanese flounder [9], Chinese clearhead icefish [10] and Northern snakehead [11]. We are also in charge of the China Auqatic 10-100-1,000 Genomics Program [12], in which ~100 fish genomes are sequencing targets for the next 3~5 years. Based on our previous experience on fish genomic studies, here we outline recent advances in related bioinformatics for fish genomics to share with public readers. Since the basic informatics includes genome assembly, genome annotation and evolutionary analysis, we discuss them one by one in this order.

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September 21, 2019  |  

Identification of a novel RASD1 somatic mutation in a USP8-mutated corticotroph adenoma.

Cushing’s disease (CD) is caused by pituitary corticotroph adenomas that secrete excess adrenocorticotropic hormone (ACTH). In these tumors, somatic mutations in the gene USP8 have been identified as recurrent and pathogenic and are the sole known molecular driver for CD. Although other somatic mutations were reported in these studies, their contribution to the pathogenesis of CD remains unexplored. No molecular drivers have been established for a large proportion of CD cases and tumor heterogeneity has not yet been investigated using genomics methods. Also, even in USP8-mutant tumors, a possibility may exist of additional contributing mutations, following a paradigm from other neoplasm types where multiple somatic alterations contribute to neoplastic transformation. The current study utilizes whole-exome discovery sequencing on the Illumina platform, followed by targeted amplicon-validation sequencing on the Pacific Biosciences platform, to interrogate the somatic mutation landscape in a corticotroph adenoma resected from a CD patient. In this USP8-mutated tumor, we identified an interesting somatic mutation in the gene RASD1, which is a component of the corticotropin-releasing hormone receptor signaling system. This finding may provide insight into a novel mechanism involving loss of feedback control to the corticotropin-releasing hormone receptor and subsequent deregulation of ACTH production in corticotroph tumors.

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